Methods and compositions for regenerating connective tissue

ABSTRACT

Connective tissue regenerative compositions and methods of repairing and regenerating connective tissue using such compositions are provided. The compositions generally comprise a bioactive hydrogel matrix comprising a polypeptide, such as gelatin, and a long chain carbohydrate, such as dextran. The hydrogel matrix may further include polar amino acids, as well as additional beneficial additives. Advantageously, the compositions include further components, such as osteoinductive or osteoconductive materials, medicaments, stem or progenitor cells, and three-dimensional structural frameworks. The compositions are useful for regenerating connective tissue, and can be administered to an area having injury to, or a loss of, connective tissue, such as bone, cartilage, tendon, and ligament.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a division of U.S. patent application Ser.No. 12/039,214, filed Feb. 28, 2008, which is a continuation of U.S.patent application Ser. No. 10/971,544, filed Oct. 22, 2004, whichclaims the benefit of U.S. Provisional Patent Application Ser. No.60/513,392, filed Oct. 22, 2003, all of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention is related to methods and compositions forregenerating connective tissue, such as bone, cartilage, ligament,tendon, and the like. In particular, the invention is related to methodsfor regenerating connective tissue through application of a hydrogelmatrix, wherein the matrix is comprised of a polypeptide, such asgelatin, and a long chain carbohydrate, such as dextran.

BACKGROUND OF THE INVENTION

Injuries to bone, such as partial or complete fracture, can be slow toheal, but such injuries generally heal on their own accord with externalimmobilization as needed, such as by applying a cast to the affectedarea. In more severe cases, more aggressive internal immobilization,such as permanently reconnecting the fractured bone with screws and/ormetal plates, may be required. Regeneration of bone tissue over therelatively short distances generally present in bone fracture readilyoccurs in most healthy patients. Bone injuries beyond simple fractures,however, present greater challenges in treatment. Long segmentaldiaphyseal bone loss, for example, can result from multiple causesincluding high-energy trauma, such as blast injury, disease, such asosteomyelitis or osteonecrosis, or wide excision of malignantconditions, such as osteosarcoma. Such conditions often result incavitation of the bone or complete loss of bone tissue across anextended length of the bone (i.e., a critical bone defect). Boneregeneration in these cases becomes increasingly challenging andsometimes impossible.

Many techniques have been used in an attempt to enhance bone growth.Most commonly, an attempt is made to replace the lost bone. Examples ofsuch techniques include autologous vascularized bone grafts, massiveallograft (generally from cadaver), and use of reabsorbable andnon-reabsorbable artificial bone. Another method for promoting boneregeneration is through the introduction of osteoinductive bioactivefactors, such as bone morphogenetic proteins (BMPs), platelet richplasma (PRP), synthetic peptides, such as P-15 (Pepgen P-15™, DentsplyInternational, York, Pa.), and bone marrow aspirates. Such bioactivefactors can be introduced into the area of bone loss through variousvehicles. Mechanical methods, such as distraction osteogenesis, are alsoemployed for promoting bone regeneration. Distraction osteogenesis is aprocess involving gradual, controlled displacement of surgically createdfractures resulting in simultaneous expansion of soft tissue and bonevolume.

A somewhat less invasive technique that is used most commonly forregenerating bone around teeth is known as “guided bone regeneration.”As the tissue surrounding a bone almost always heals faster than thebone itself, the faster-healing tissue often expands into and fills thespace where the bone is missing, hindering the bone regeneration. Inguided bone regeneration, a biocompatible membrane is placed between thetissue and the bone acting as a barrier, which prevents growth of thetissue into the bone. Often, a bone graft is inserted under the barrier.The membranes are typically designed to dissolve away after severalweeks.

A variation on this procedure is known as “protected bone regeneration”and is based on the theory that three prerequisites for bone healing arerequired: 1) adequate blood supply, 2) abundant bone forming cells, and3) protected healing space. See, Holmes, R. E., Lemperle, S. M., andCalhoun, C. J., “Protected Bone Regeneration,” Scientific Data Series inResorbable Fixation, distributed by Medtronic Sofamor Danek, availableon-line at http://www.macropore.com/pdf/Protected_Bone.pdf. Adequateblood supply is a known requirement for bone regeneration as it suppliesthe necessary oxygen and nutrients, as well as mesenchymal stem cells(the bone forming cells). As described above, the healing space of thebone must also be protected from the ingrowth of surrounding tissue.According to the above-noted publication, all of the statedprerequisites can be met through the use of a reabsorbable polymerprotective sheet offering a physiologically balanced porosity forpositive cellular exchange and the opportunity for vascularinfiltration, while preventing interposition of adjacent soft tissues.

While there are several methods currently known, treatment of injuryresulting in major bone loss remains a difficult clinical problem.Furthermore, approximately 10% of all long bone fractures are non-unionfractures that do not heal spontaneously. Thus, there remains a need formethods for bone regeneration that are effective at promoting bonetissue growth and that are as non-invasive as possible.

SUMMARY OF THE INVENTION

It has been discovered that the matrix described herein is capable ofsuccessfully promoting regeneration of connective tissue. Surprisingly,the matrix is even useful for effecting bone regeneration in bone withdefects that will not normally spontaneously heal. The present inventionprovides a method for connective tissue regeneration comprisingadministration of a bioactive hydrogel matrix into the site in need ofconnective tissue regeneration. As used herein, “bioactive” is intendedto indicate the ability to facilitate a cellular or tissue response,such as, induction of vasculogenesis, promotion of cellular attachmentto a scaffold material, and promotion of tissue regeneration.

In one aspect of the invention, there is provided a method forregenerating connective tissue. In one embodiment, the method comprisesadministering to a site in need of connective tissue regeneration abioactive hydrogel matrix comprising a polypeptide and a long chaincarbohydrate. The polypeptide can be selected from tissue-derivedpolypeptides or synthetic polypeptides. In one embodiment, thepolypeptide is skin-derived gelatin. In another embodiment, thepolypeptide is bone-derived gelatin. Exemplary long chain carbohydratesinclude polysaccharides and sulfated polysaccharides. In one embodiment,the long chain carbohydrate is dextran. The bioactive hydrogel matrixfurther comprises one or more components selected from the groupconsisting of polar amino acids, polar amino acid analogues orderivatives and divalent cation chelators, such asethylenediaminetetraacetic acid (EDTA) or salts thereof.

The bioactive hydrogel matrix, as used in the above method, can furtherinclude one or more various structuring agents, medicaments, or otheragents useful for facilitating or mediating connective tissueregeneration.

In one embodiment of the invention, the bioactive hydrogel matrix canfurther comprise at least one osteoinductive or osteoconductivematerial. In this embodiment of the invention, the method isparticularly useful for regenerating bone; moreover, the use ofosteoinductive or osteoconductive materials is not limited to boneregeneration.

In yet another embodiment of the invention, the bioactive hydrogelmatrix further comprises at least one medicament. Any medicamentrecognizable by one of skill in the art as useful in the treatment ofconnective tissue injury, particularly in methods of regeneratingconnective tissue, could be used. For example, the medicaments caninclude antivirals, antibacterials, anti-inflammatories,immunosuppresants, analgesics, anticoagulants, or various wound healingpromotion agents.

In one particular embodiment of the invention, the bioactive hydrogelmatrix further comprises stem or progenitor cells, such asadipose-derived adult stem (ADAS) cells or mesenchymal stem cells. Suchcells are known in the art as useful in various therapies due to theirability to differentiate into a number of cell types. ADAS cells inparticular are known to differentiate into cell types includingchondrocytes and osteoblasts.

In still another embodiment of the method of the invention, thebioactive hydrogel matrix is at least partially contained within athree-dimensional structural framework. Accordingly, the structuralframework can be included with the bioactive hydrogel matrix prior toadministration of the bioactive hydrogel matrix to the site in need ofconnective tissue regeneration. Alternatively, the structural frameworkcan be formed around the site in need of connective tissue regenerationat the time of administration of the bioactive hydrogel matrix (i.e.,formed shortly before or shortly after administration of the bioactivehydrogel matrix). The three-dimensional structural framework, therefore,includes any material capable of providing load-bearing structuralsupport or anatomical space for cellular infiltration and includes, forexample, a metal cage, a sintered ceramic framework, a collagen sponge,or allogenic or autologous bone. The structural framework can furtherinclude three dimensional structures prepared from polymeric materials,including biopolymers.

The bioactive hydrogel matrix can also be used in the method of theinvention in a dehydrated form. In such form, the bioactive hydrogelmatrix retains its beneficial properties yet can be stored andtransported in a solid form, being capable of re-hydration for use inthe method of the present invention. In one embodiment, the bioactivehydrogel matrix is administered in a dehydrated form such that bodyfluids re-hydrate the bioactive hydrogel matrix. In another embodiment,the bioactive hydrogel matrix is in dehydrated form and the methodfurther comprises re-hydrating the bioactive hydrogel matrix with are-hydrating fluid prior to administering the bioactive hydrogel matrixto the site in need of connective tissue regeneration. In dehydratedform, the bioactive hydrogel matrix can be shaped or processed into avariety of shapes and forms. For example, the dehydrated bioactivehydrogel matrix can be in a unitary piece capable of being shaped toprecisely fit the site in need of connective tissue regeneration.Alternately, the dehydrated bioactive hydrogel matrix can be inparticulate form. The particulate dehydrated bioactive hydrogel matrixcould be mixed into a solution containing other beneficial ingredients,such as stem or progenitor cells or medicaments, combined withosteoinductive or osteoconductive materials to form a putty orpaste-like material for placement into the site in need of connectivetissue regeneration, or used in other preparations that would be usefulin the method of the invention.

In other embodiments of the invention, it may be useful for thebioactive hydrogel matrix to have additional structure or strength inthe absence of additives. Accordingly, the present invention furtherencompasses embodiments wherein the bioactive hydrogel matrix is incrosslinked form, the long chain carbohydrate being covalentlycrosslinked to the polypeptide. In such embodiments, the bioactivehydrogel matrix can be used alone in the method of the invention or maybe used in conjunction with other components as described herein.

In one embodiment of the invention, the bioactive hydrogel matrix isinserted into an area of a bone in need of repair or regeneration (i.e.,a bone defect). The amount of the bioactive hydrogel matrix used in thebone can vary depending upon the size of the bone defect, the form ofthe bioactive hydrogel matrix, and the presence or absence of additivesas described herein. Typically, the total amount of the bioactivehydrogel matrix used is the amount required to fill the area of boneloss.

According to another embodiment of the present invention, the hydrogelmatrix can be used for repair of soft tissue either separately or inconjunction with regeneration of nearby hard tissue, such as bone.According to this embodiment, the bioactive hydrogel matrix isadministered around and/or injected into the soft tissue.

According to another embodiment of the present invention, the hydrogelmatrix can be used for repair and/or regeneration of non-bone connectivetissue. According to this embodiment, the bioactive hydrogel matrix isadministered to an area having loss of, or damage to, connective tissue,which includes tissue arising from fibroblasts, such as tendon andligament, or chondrocytes, such as cartilage.

According to another aspect of the present invention, there are providedvarious connective tissue regenerative compositions. The compositionsare particularly useful in the regeneration of connective tissue or fortreatment of patients having various connective tissue degenerativediseases. Accordingly, the compositions described herein areparticularly useful in the methods of the invention also describedherein.

In one embodiment of this aspect of the invention, the connective tissueregenerative composition comprises a three-dimensional structuralframework and a bioactive hydrogel matrix at least partially containedwithin the three-dimensional structural framework, wherein the bioactivehydrogel matrix comprises a polypeptide and a long chain carbohydrate.The bioactive hydrogel matrix preferably further comprises one or morecomponents selected from the group consisting of polar amino acids,polar amino acid analogues or derivatives, and divalent cationchelators, such as EDTA or salts thereof. In one particular embodiment,the three-dimensional structural framework includes a crosslinkedhydrogel matrix. In another preferred embodiment, the three-dimensionalstructural framework includes a collage sponge.

In another embodiment, the connective tissue regenerative compositioncomprises at least one osteoinductive or osteoconductive material and abioactive hydrogel matrix comprising a polypeptide and a long chaincarbohydrate. The osteoinductive or osteoconductive material can bedispersed within the bioactive hydrogel matrix. In one preferredembodiment, the osteoinductive or osteoconductive material and thebioactive hydrogel matrix can be in admixture. The bioactive hydrogelmatrix can be in a hydrated form or can be in a dehydrated form.

In still another embodiment of the invention, the connective tissueregenerative composition comprises stem or progenitor cells and abioactive hydrogel matrix comprising a polypeptide and a long chaincarbohydrate. Again, the bioactive hydrogel matrix can be in a hydratedform or can be in a dehydrated form.

According to another aspect of the present invention, the bioactivehydrogel matrix can be used for attaching or reattaching two or moreconnective tissues. In one embodiment of this aspect of the invention,the method comprises: coating at least a portion of at least one of afirst and second connective tissue with a bioactive hydrogel matrixcomprising a polypeptide and a long chain carbohydrate; contacting thefirst connective tissue to the second connective tissue at a point ofattachment; and attaching the first connective tissue to the secondconnective tissue using sutures, staples or other appropriate means.Such a method is particularly useful for attaching connective tissue,such as tendon or ligament, to bone. The method is further useful forattaching soft connective tissue to other soft connective tissue, suchas tendon to tendon or ligament to ligament.

According to another aspect of the invention, the bioactive hydrogelmatrix is used in a method for treating degenerative diseases of thenatural joint of a patient in need of treatment thereof. In oneembodiment, the method comprises: applying to a joint affected by adegenerative disease, a bioactive hydrogel matrix comprising apolypeptide and a long chain carbohydrate. Further, optionally, thebioactive hydrogel matrix can include stem or progenitor cells.Preferentially, the administering step comprises injecting the bioactivehydrogel matrix into the affected joint. The method is particularlyuseful for halting progression of or reversing degenerative jointdiseases, such as osteoarthritis.

The compositions and methods of the present invention are particularlyuseful for repairing connective tissue of the knee, such as the anteriorcruciate ligament, the posterior cruciate ligament, the patellar tendon,the quadriceps tendon, and the anterior meniscofemoral ligament.

The compositions and methods of the invention are further useful fortreating a patient having an artificial joint. In particular, theconnective tissue regenerative compositions can be administered aroundthe site of the artificial joint, either during placement of theartificial joint or post-surgery, to facilitate integration of theartificial joint into the surrounding tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described the invention in general terms, reference will nowbe made to the accompanying drawings, wherein:

FIG. 1 illustrates formation of open alpha chains derived from collagenmonomers;

FIGS. 2A and 2B illustrate the effect of the association of thecollagen-derived alpha chains with dextran;

FIG. 3 illustrates the effect of other additives used in the bioactivehydrogel matrix of the invention;

FIG. 4 graphically illustrates cellular aggregation across various celltypes in the presence of the bioactive hydrogel matrix of the presentinvention;

FIG. 5 illustrates the effect of the bioactive hydrogel matrix of thepresent invention on the expression of the BMP-2 gene as compared toexpression in cells in serum free medium (SFM);

FIG. 6 illustrates the increased expression of connective tissue growthfactor (CTGF) messenger RNA in chondrosarcoma cells treated with thebioactive hydrogel matrix of the invention as compared to cells in SFM;

FIG. 7 illustrates the expression of aggrecan messenger RNA inchondrosarcoma cells treated with the bioactive hydrogel matrix of theinvention compared to cells in SFM;

FIG. 8 illustrates a crosslinked bioactive hydrogel matrix of theinvention comprising dextran and gelatin;

FIG. 9 illustrates the effect of the bioactive hydrogel matrix of thepresent invention on the production of BMP-2 protein as compared toproduction in cells in serum containing medium (SCM); and

FIG. 10 illustrates the effect of the crosslinked bioactive hydrogelmatrix of the present invention on the expression of the BMP-2 gene.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. This invention may, however, be embodied inmany different forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

The formulation of a thermoreversible hydrogel matrix providing a cellculture medium and composition for preserving cell viability is taughtby U.S. Pat. No. 6,231,881, herein incorporated by reference in itsentirety. Additionally, a hydrogel matrix useful in promotingvascularization is provided in U.S. Pat. No. 6,261,587, hereinincorporated by reference in its entirety. The thermoreversible hydrogelmatrix taught by these references is a gel at storage temperatures andmolten at physiologic temperatures, and comprises a combination of acollagen-derived component, such as gelatin, a long chain carbohydrate,such as dextran, and effective amounts of other components, such aspolar amino acids.

The present invention provides connective tissue regenerativecompositions and methods of regenerating connective tissue at a site inneed of connective tissue regeneration. The compositions and method ofthe invention include a bioactive hydrogel matrix generally comprising apolypeptide and a long chain carbohydrate.

A polypeptide, as used herein, is intended to encompass anytissue-derived or synthetically produced polypeptide, such as collagensor collagen-derived gelatins. Although collagen-derived gelatin is thepreferred polypeptide component, other gelatin-like componentscharacterized by a backbone comprised of sequences of amino acids havingpolar groups that are capable of interacting with other molecules can beused. For example, keratin, decorin, aggrecan, glycoproteins (includingproteoglycans), and the like could be used to provide the polypeptidecomponent. In one embodiment, the polypeptide component is porcinegelatin from partially hydrolyzed collagen derived from skin tissue.Polypeptides derived from other types of tissue could also be used.Examples include, but are not limited to, tissue extracts from arteries,vocal chords, pleura, trachea, bronchi, pulmonary alveolar septa,ligaments, auricular cartilage or abdominal fascia; the reticularnetwork of the liver; the basement membrane of the kidney; or theneurilemma, arachnoid, dura mater or pia mater of the nervous system.Purified polypeptides including, but not limited to, laminin, nidogen,fibulin, and fibrillin or protein mixtures such as those described byU.S. Pat. No. 6,264,992 and U.S. Pat. No. 4,829,000, extracts from cellculture broth as described by U.S. Pat. No. 6,284,284, submucosaltissues such as those described in U.S. Pat. No. 6,264,992, or geneproducts such as described by U.S. Pat. No. 6,303,765 may also be used.Another example of a suitable polypeptide is a fusion protein formed bygenetically engineering a known reactive species onto a protein.

The polypeptide component preferably has a molecular mass range of about3,000 to about 3,000,000 Da, more preferably about 30,000 to about300,000 Da, most preferably about 50,000 to about 250,000 Da. Molecularmass can be expressed as a weight average molecular mass (M_(w)) or anumber average molecular mass (M_(n)). Both expressions are based uponthe characterization of macromolecular solute containing solution ashaving an average number of molecules (n_(i)) and a molar mass for eachmolecule (M_(i)). Accordingly, number average molecular mass is definedby formula 1 below.

$\begin{matrix}{M_{n} = \frac{\sum{n_{i}M_{i}}}{\sum n_{i}}} & (1)\end{matrix}$

Weight average molecular mass (also known as molecular mass average) isdirectly measurable using light scattering methods and is defined byformula 2 below.

$\begin{matrix}{M_{w} = \frac{\sum{n_{i}M_{i}^{2}}}{\sum{n_{i}M_{i}}}} & (2)\end{matrix}$

Molecular mass can also be expressed as a Z-average molar mass (M_(z)),wherein the calculation places greater emphasis on molecules with largemolar masses. Z-average molar mass is defined by formula 3 below.

$\begin{matrix}{M_{z} = \frac{\sum{n_{i}M_{i}^{3}}}{\sum{n_{i}M_{i}^{2}}}} & (3)\end{matrix}$

Unless otherwise noted, molecular mass is expressed herein as weightaverage molecular mass.

In addition to molecular mass, polymer solutions can also be physicallydescribed in terms of polydispersity, which represents the broadness ofthe molecular mass distribution within the solution, such distributionbeing the range of different molecular masses of the individual polymermolecules in the solution. Polydispersity is the ratio of the numberaverage molecular mass to the weight average molecular mass, which isdefined by formula 4 below.

$\begin{matrix}{{Polydispersity} = \frac{M_{w}}{M_{n}}} & (4)\end{matrix}$

If polydispersity is equal to 1 (i.e., M_(n) equals M_(w)), the polymeris said to be monodisperse. A truly monodisperse polymer is one whereall polymer molecules within the solution are of a single, identicalmolecular mass. As M_(n) changes with M_(w), the polydispersity changes,always being greater than 1. The polydispersity of a given polymersolution can affect the physical characteristics of the polymer, and,therefore, the interaction of the polymer with another polymer. Researchhas shown that in aqueous mixtures of biopolymers (including gelatin anddextran), an increase in molecular weight results in a less compatiblesystem with a higher phase separation temperature, whereas a decrease inconcentration results in a more compatible system with a lower phaseseparation temperature (see E. H. A. de Hoog and R. H. Tromp, On thephase separation kinetics of an aqueous biopolymer mixture in thepresence of gelation: the effect of the quench depth and the effect ofthe molar mass, Colloids and Surfaces A: Physicochemical and EngineeringAspects, 213 (2-3), Pages 221-234). Preferably, the polypeptide usedaccording to the present invention has a polydispersity close to 1. Inone preferred embodiment, the polypeptide has a polydispersity of 1 toabout 4, more preferably, about 1 to about 3, most preferably about 1.1to about 2.4.

The polypeptide used in the bioactive hydrogel matrix of the inventionis preferably a gelatin, such as collagen derived gelatin.

Collagen is a major protein component of the extracellular matrix ofanimals. Early in fetal development, a more open form of collagen(compared to tightly bound mature collagen) is associated with largecarbohydrate molecules, and serves as the predominant tissuescaffolding. It is believed that attachment of differentiated orincompletely differentiated cells of mesenchymal origin to this polar,proteoglycan-like, collagen scaffolding results in a specific hosttissue response. This response is to guide the differentiation ofmesenchymal tissue.

Collagen is assembled into a complex fibrillar organization. The fibrilsare assembled into bundles that form the fibers. The fibrils are made offive microfibrils placed in a staggered arrangement. Each microfibril isa collection of collagen rods. Each collagen rod is a right-handedtriple-helix, each strand being itself a left-handed helix. Collagenfibrils are strengthened by covalent intra- and intermolecularcross-links which make the tissues of mature animals insoluble in coldwater. When suitable treatments are used, collagen rods are extractedand solubilized where they keep their conformation as triple-helices.This is denatured collagen and differs from the native form of collagen,but has not undergone sufficient thermal or chemical treatment to breakthe intramolecular stabilizing covalent bonds found in collagen. Whencollagen solutions are extensively heated, or when the native collagencontaining tissues are subjected to chemical and thermal treatments, thehydrogen and covalent bonds that stabilize the collagen helices arebroken, and the molecules adopt a disordered conformation. By breakingthese hydrogen bonds, the polar amine and carboxylic acid groups are nowavailable for binding to polar groups from other sources or themselves.This material is gelatin and is water-soluble at 40-45° C.

As noted above, gelatin is a form of denatured collagen, and is obtainedby the partial hydrolysis of collagen derived from the skin, whiteconnective tissue, or bones of animals. Gelatin may be derived from anacid-treated precursor or an alkali-treated precursor. Gelatin derivedfrom an acid-treated precursor is known as Type A, and gelatin derivedfrom an alkali-treated precursor is known as Type B. The macromolecularstructural changes associated with collagen degradation are basicallythe same for chemical and partial thermal hydrolysis. In the case ofthermal and acid-catalyzed degradation, hydrolytic cleavage predominateswithin individual collagen chains. In alkaline hydrolysis, cleavage ofinter- and intramolecular cross-links predominates.

Preferably, the gelatin used in the present invention is skin-derivedgelatin or bone derived gelatin. In one preferred embodiment, thegelatin has a molecular mass of about 80,000 Da to about 200,000 Da.Further, it is preferred that the gelatin have a polydispersity of 1 toabout 3. In one preferred embodiment, the gelatin has a polydispersityof about 1.1 to about 2.4.

The polypeptide, such as gelatin, is preferentially present at aconcentration of about 0.01 to about 40 mM, preferably about 0.05 toabout 30 mM, most preferably about 0.25 to about 5 mM. Advantageously,the gelatin concentration is approximately 0.75 mM. The aboveconcentrations provide a non-flowable phase at storage temperature(below about 33° C.) and a flowable phase at treatment temperature(about 35 to about 40° C.).

The bioactive hydrogel matrix of the present invention also comprises along chain carbohydrate. The phrase long chain carbohydrate is generallyintended to encompass any polysaccharide or sulfated polysaccharideconsisting of more than about 10 monosaccharide residues joined to eachother by glycosidic linkages. The phrase is also intended to encompassother long chain carbohydrates, including heterosaccharides, andspecific classes of carbohydrates, such as starches, sugars, celluloses,and gums. The long chain carbohydrate may consist of the samemonosaccharide residues, or various monosaccharide residues orderivatives of monosaccharide residues. Dextran, a preferredpolysaccharide, solely comprises glucose residues.

Any polysaccharide, including glycosaminoglycans (GAGs) orglucosaminoglycans, with suitable viscosity, molecular mass and otherdesirable properties may be utilized in the present invention. Byglycosaminoglycan is intended any glycan (i.e., polysaccharide)comprising an unbranched polysaccharide chain with a repeatingdisaccharide unit, one of which is always an amino sugar. Thesecompounds as a class carry a high negative charge, are stronglyhydrophilic, and are commonly called mucopolysaccharides. This group ofpolysaccharides includes heparin, heparan sulfate, chondroitin sulfate,dermatan sulfate, keratan sulfate, and hyaluronic acid. These GAGs arepredominantly found on cell surfaces and in the extracellular matrix. Byglucosaminoglycan is intended any glycan (i.e. polysaccharide)containing predominantly monosaccharide derivatives in which analcoholic hydroxyl group has been replaced by an amino group or otherfunctional group such as sulfate or phosphate. An example of aglucosaminoglycan is poly-N-acetyl glucosaminoglycan, commonly referredto as chitosan. Exemplary polysaccharides that may be useful in thepresent invention include dextran, heparan, heparin, hyaluronic acid,alginate, agarose, carageenan, amylopectin, amylose, glycogen, starch,cellulose, chitin, chitosan and various sulfated polysaccharides such asheparan sulfate, chondroitin sulfate, dextran sulfate, dermatan sulfate,or keratan sulfate.

The long chain carbohydrate preferably has a molecular mass of about2,000 to about 8,000,000 Da, more preferably about 20,000 to about1,000,000 Da, most preferably about 200,000 to about 800,000 Da. In oneembodiment, the long chain carbohydrate has a molecular mass ofapproximately 500,000 Da.

Preferably, the long chain carbohydrate used according to the presentinvention has a polydispersity close to 1. In one preferred embodiment,the polypeptide has a polydispersity of 1 to about 3, more preferably,about 1.1 to about 2.4.

As previously noted, one preferred long chain carbohydrate for use inthe present invention is dextran. Dextran typically comprises linearchains of α(1→6)-linked D-glucose residues, often with α(1→2)- orα(1→3)-branches. Native dextran, produced by a number of species ofbacteria of the family Lactobacilliaceae, is a polydisperse mixture ofcomponents. Dextrans have been widely used as plasma substitutes andblood extenders, are considered fully biocompatible, and aremetabolizable. Dextrans are available in a wide range of averagemolecular masses, varying from about 4,000 to about 40,000,000 Da.Preferably, the dextran used in the invention has a molecular mass ofabout 200,000 to about 800,000 Da, most preferably about 300,000 toabout 600,000 Da. In one preferred embodiment, the dextran has amolecular mass of approximately 500,000 Da. Dextrans have varying ratesof resorption in vivo from about two to about 20 days depending on theirmolecular mass.

The long chain carbohydrate, such as dextran, is preferentially presentat a concentration of about 0.01 to about 10 mM, preferably about 0.01to about 1 mM, most preferably about 0.01 to about 0.5 mM. In oneembodiment, dextran is present at a concentration of about 0.1 mM.

While native dextran is generally used in the present invention, the useof dextran derivatives, such as dextran sulfate and dextran phosphate isalso within the scope of the invention. In one embodiment, thederivatives are free radical polymerizable, preferablyphotopolymerizable derivatives, such as acrylates. According to thisembodiment, the composition can be injected as a viscous liquid andpolymerized in situ to form a solid material. The dextran can also beselected to degrade at a rate which approximates ingrowth of new bone ortissue. Those compositions that include free radical polymerizablegroups may also include polymerization initiators, such asphotoinitiators, such as benzoin ethers, and thermally activatableinitiators, such as azobisisobutyronitrile (AIBN) and di-t-butyl ether.Free radical polymerization initiators, and conditions for carrying outfree radical polymerizations, are well known to those of skill in theart, and any of such methods are encompassed by the present invention.

In a preferred embodiment, gelatin and dextran are components of thebioactive hydrogel matrix of the present invention. For ease ofdescribing the invention, the terms “gelatin” and “dextran” are usedthroughout with the understanding that various alternatives as describedabove, such as other polypeptides and other long chain carbohydratesreadily envisioned by those skilled in the art, are contemplated by thepresent invention.

Although not bound by any particular theory, the present invention isintended to provide a matrix scaffolding designed to maximize the polaramino acid hydrogen bonding sites found in alpha chains derived fromcollagen. These alpha chains, or gelatin, are preferably derived frompig gelatin, and stabilized by 500,000 Da molecular mass dextran, orother long chain carbohydrates, added while the alpha chains are heated.The positively charged polar groups of the collagen-derived alpha chainsare then able to associate with the negatively charged —OH groups of therepeating glucose units found in the dextran. The gelatin and thedextran form a proteoglycan-type structure. FIGS. 1-3 illustrate theinteraction between the various components of the preferred embodimentof the matrix of the invention and interaction between the matrix andthe tissue of a patient.

FIG. 1 illustrates the creation of polar alpha chains 15 fromtropocollagen 10 derived from mature collagen. Heating tropocollagen 10disrupts the hydrogen bonds that tightly contain the triple strandedmonomers in mature collagen. By breaking these hydrogen bonds, the polaramine and carboxylic acid groups are now available for binding to polargroups from other sources or themselves.

FIGS. 2A-2B illustrate stabilization of the matrix monomeric scaffoldingby the introduction of a long chain carbohydrate 20, such as dextran. Asshown in FIG. 2B, without the long chain carbohydrate 20, the alphachain 15 will form hydrogen bonds between the amino and carboxylic acidgroups within the linear portion of the monomer and fold upon itself,thus limiting available sites for cellular attachment. As depicted inFIG. 2A, the long chain carbohydrate 20 serves to hold the alpha chain15 open by interfering with this folding process.

In addition to the polypeptide and long chain carbohydrate, thebioactive hydrogel matrix can further comprise one or more componentsuseful for enhancing the bioadhesiveness of the hydro gel matrix.Examples of such components include polar amino acids, polar amino acidanalogues or derivatives, divalent cation chelators, and combinationsthereof. In one preferred embodiment, all of the bioactive hydrogelmatrix ingredients are provided in admixture.

The bioactive hydrogel matrix preferably includes one or more polaramino acids in an effective amount to increase the rigidity of thehydrogel matrix and allow direct administration of the hydrogel matrix,such as through injection, to a site in need of connective tissueregeneration. As used herein, polar amino acids are commonly defined andintended to include tyrosine, cysteine, serine, threonine, asparagine,glutamine, asparatic acid, glutamic acid, arginine, lysine, andhistidine. Peferentially, the amino acids are selected from the groupconsisting of cysteine, arginine, lysine, histidine, glutamic acid,aspartic acid. When polar amino acids are present in the bioactivehydrogel matrix, the polar amino acids are preferentially present in aconcentration of about 3 to about 150 mM, preferably about 10 to about65 mM, and more preferably about 15 to about 40 mM.

Advantageously, the added polar amino acids comprise L-glutamic acid,L-lysine, and L-arginine. The final concentration of L-glutamic acid isgenerally about 2 to about 60 mM, preferably about 5 to about 40 mM,most preferably about 10 to about 30 mM. In one embodiment, theconcentration of L-glutamic acid is about 20 mM. The final concentrationof L-lysine is generally about 0.5 to about 30 mM, preferably about 1 toabout 15 mM, most preferably about 1 to about 10 mM. In one embodiment,the concentration of L-lysine is about 5.0 mM. The final concentrationof L-arginine is generally about 1 to about 40 mM, preferably about 1 toabout 30 mM, most preferably about 5 to about 20 mM. In one embodiment,the final concentration of arginine is about 15 mM.

By amino acid is intended all naturally occurring alpha amino acids inboth their D and L stereoisomeric forms, and their analogues andderivatives. An analog is defined as a substitution of an atom orfunctional group in the amino acid with a different atom or functionalgroup that usually has similar properties. A derivative is defined as anamino acid that has another molecule or atom attached to it. Derivativeswould include, for example, acetylation of an amino group, amination ofa carboxyl group, or oxidation of the sulfur residues of two cysteinemolecules to form cystine. As previously noted, the bioactive hydrogelmatrix of the invention can include one or more polar amino acidanalogues or derivatives.

Amino acids used in the bioactive hydrogel matrix of the presentinvention can also be present as dipeptides, which are particularbeneficial for delivery of amino acids having decreased watersolubility, such as L-glutamine. Accordingly, amino acids added to thehydrogel matrix can include dipeptides, such as L-alanyl-L-glutamine.When present in the hydrogel matrix, the concentration range forL-alanyl-L-glutamine is preferably about 0.001 to about 1 mM, morepreferably about 0.005 to about 0.5 mM, most preferably about 0.008 toabout 0.1 mM. In one particular embodiment, the final concentration ofL-alanyl-L-glutamine is about 0.01 mM.

The added amino acids can also include L-cysteine, which is advantageousin many regards. Cysteine is useful for providing disulfide bridges,further adding support and structure to the bioactive hydrogel matrixand increasing its resistance to force. The final concentration ofL-cysteine is generally about 5 to about 5000 μM, preferably about 10 toabout 1000 μM, most preferably about 100 to about 1000 μM. In oneembodiment, the final concentration of cysteine is about 700 μM.L-cysteine also acts as a nitric oxide scavenger or inhibitor. Nitricoxide inhibitors include any composition or agent that inhibits theproduction of nitric oxide or scavenges or removes existing nitricoxide. Nitric oxide, a pleiotropic mediator of inflammation, is asoluble gas produced by endothelial cells, macrophages, and specificneurons in the brain, and is active in inducing an inflammatoryresponse. Nitric oxide and its metabolites are known to cause cellulardeath from nuclear destruction and related injuries.

Accordingly, the bioactive hydrogel matrix can optionally include one ormore additional nitric oxide inhibitors, such as aminoguanidine,N-monomethyl-L-arginine, N-nitro-L-arginine, cysteine, heparin, andmixtures thereof. When present in the hydrogel matrix, the finalconcentration of nitric oxide inhibitors is generally about 5 to about500 μM, preferably about 10 to about 100 μM, most preferably about 15 toabout 25 μM. In one embodiment, the final concentration is about 20 μM.

Advantageously, intact collagen can be optionally added to the bioactivehydrogel matrix to provide an additional binding network and provideadditional support to the matrix. The final concentration of the intactcollagen present in the hydrogel matrix is from about 0 to about 5 mM,preferably about 0 to about 2 mM, most preferably about 0.05 to about0.5 mM.

Additionally, the bioactive hydrogel matrix may optionally include oneor more divalent cation chelators, which increase the rigidity of thematrix by forming coordinated complexes with any divalent metal ionspresent. The formation of such complexes leads to the increased rigidityof the matrix by removing the inhibition of hydrogen bonding between—NH₂ and —COOH caused by the presence of the divalent metal ions. Apreferred example of a divalent cation chelator that is useful in thepresent invention is ethylenediaminetetraacetic acid (EDTA) or a saltthereof. The concentration range for the divalent cation chelator, suchas EDTA, is generally about 0.01 to about 10 mM, preferably 1 to about 8mM, most preferably about 2 to about 6 mM. In a one embodiment, EDTA ispresent at a concentration of about 4 mM.

EDTA is also an example of another group of compounds useful asadditives for the bioactive hydrogel matrix, superoxide inhibitors.Superoxide is a highly toxic reactive oxygen species, whose formation iscatalyzed by divalent transition metals, such as iron, manganese,cobalt, and sometimes calcium. Highly reactive oxygen species such assuperoxide (O₂ ⁻) can be further converted to the highly toxic hydroxylradical (OH⁻) in the presence of iron. By chelating these metalcatalysts, EDTA serves as an antioxidant. Accordingly, the bioactivehydrogel matrix can include one or more superoxide inhibitor.

Optionally, trace mineral nutrients and salts thereof, such as zincsulfate, can be added to the bioactive hydrogel matrix. Zinc hasbeneficial wound healing effects that are particularly useful in thepresent invention. When present in the hydrogel matrix, theconcentration range for zinc is generally about 0.005 mM to about 3 mM,preferably about 0.01 to about 2 mM, most preferably about 0.02 to about1 mM. In one particular embodiment, the final concentration of zinc isabout 0.03 mM.

The bioactive hydrogel matrix is preferably based upon a physiologicallycompatible buffer, one embodiment being Medium 199, a common nutrientsolution used for in vitro culture of various mammalian cell types(available commercially from Sigma Chemical Company, St. Louis, Mo.).The buffer can be further supplemented with additives and additionalamounts of some medium components, such as supplemental amounts of polaramino acids as described above.

The bioactive hydrogel matrix can also be formulated in other bufferedsolutions, including buffered solutions regarded as simplified inrelation to Medium 199. For example, a phosphate buffer formulated toyield physiological osmotic pressures after hydrogel matrix compoundingcan be prepared using 1.80 mM KH₂PO₄ and 63 mM Na₂HPO₄.

The bioactive hydrogel matrix of the present invention is particularlyuseful for repairing and regenerating connective tissue because of theopen structure of the hydrogel matrix and the inherent ability of thehydrogel matrix to interact with physiological material. FIG. 3illustrates the effect of polar amino acids and/or L-cysteine added tostabilize the monomer/carbohydrate units 25 by linking the exposedmonomer polar sites to, for example, arginine's amine groups or glutamicacid's carboxylic acid groups. Furthermore, disulfide linkages can beformed between L-cysteine molecules (thereby forming cystine), which inturn forms hydrogen bonds to the monomeric alpha chains 15. Thestability imparted by the polar amino acids, polar amino acid analoguesand derivatives, and intact collagen is particularly advantageous formaintaining the open structure of the gelatin and keeping the activesites available for therapeutic benefit.

The hydrogen bonds formed between these additional amino acids andmonomer/carbohydrate units 25 are broken when the matrix is liquefiedupon heating, and the polar groups are freed to attach themonomer/dextran units to exposed patient tissue surfaces. In preferredembodiments, EDTA or a salt thereof is also present to chelate divalentcations and thereby prevent divalent cations from being preferentiallyattracted to the exposed polar groups of the monomer/carbohydrate units25 to the exclusion of the polar amino acids.

Normally, the tearing of tissue secondary to trauma stimulatesproduction and release of nitric oxide, initiating recruitment of immuneand inflammatory cells that phagocytise or release chemicals to destroyforeign substances. By providing local and temporal inhibition of nitricoxide and superoxide release and production, nitric oxide inhibitors,such as aminoguanidine and cysteine, and superoxide inhibitors, such asEDTA, allow the collagen derived alpha chain/dextran units 25 to bindand become integrated on the exposed tissue surface. The alphachain/dextran units 25 then serve as the scaffolding on which formerlydifferentiated host cells de-differentiate into “mesenchymoid”morphology. This de-differentiation process is followed by integrationof these incompletely differentiated cells into host tissue. Thesemesenchymoid cells are then able to promote areas of their genome thatleads to differentiation into cell types required for tissue healing andregeneration.

By providing a proteoglycan-like scaffolding similar to that found inthe early stages of fetal development, and using structural stabilizersthat serve a secondary purpose in enhancing host response to thescaffolding upon exposure to host tissues, the matrix serves as abiocompatible device capable of increasing vascularization and promotingwound healing and local tissue regeneration, even in the case of largeareas of bone loss. Because the matrix promotes tissue-specificregeneration, as occurs during embryogenesis and fetogenesis wheresimilar types of scaffolding are present, it has now been discoveredthat the matrix of the invention can be used to successfully treat boneinjuries that are typically non-responsive to conventional treatments,such as long segmental diaphyseal bone loss, cavitation, and simplefractures in patients having abnormally low ability to regenerate bonetissue. Furthermore, it has been discovered that the bioactive hydrogelmatrix of the present invention can be used to successfully treatadditional types of injuries often known to be difficult to treat orslow to heal, such as injuries to non-bone connective tissues, such astendon, ligament, and cartilage.

In vitro testing has shown that the bioactive hydrogel matrix of theinvention exhibits a remarkable ability to bind to and hence promotecell aggregation across multiple cell types. Treatment of culturedosteoblasts (human osteosarcoma cell line SAOS-2) with the bioactivehydrogel matrix resulted in approximately 80% cellular aggregation. Inone comparative study, cells were treated with the bioactive hydrogelmatrix of the invention, and cells (control) were treated with gelatinalone. Cell types tested were fibroblasts, osteoblasts, chondrocytes,and adipocytes. The cells were stained with trypan blue and visuallyinspected. The cells treated with the bioactive hydrogel matrix wereevident as large clumps (i.e., aggregates), while the control cells(those treated with gelatin alone) were evident as single cells and notaggregated. This illustrates how the intact bioactive hydrogel matrixbinds to and aggregates cells important in wound healing, bone repair,and non-bone connective tissue repair. This binding and subsequentinteraction does not occur when only gelatin is present. Furthermore,previous similar studies with fibroblasts indicated the binding andaggregation also did not occur after treatment with dextran alone.

FIG. 4 provides quantification of the aggregation of the cells in thestudy described above. As shown in FIG. 4, after treatment with thebioactive hydrogel matrix of the invention, all four cell typesdemonstrated approximately 80% aggregation. Comparatively, the cellstreated with gelatin alone demonstrated less than 30% aggregation. Thebinding of the bioactive hydrogel matrix to cells as evidenced by theaggregation is believed to be the first key step in the action of thebioactive hydrogel matrix on cellular activity. The aggregation is aresult of the cells interacting with the open polar co-polymer structureof the bioactive hydrogel matrix.

The bioactive hydrogel matrix of the invention also exhibits additionalaction necessary for bone regeneration. In one study, treatment ofcultured osteoblasts with the bioactive hydrogel matrix of the inventionresulted in a greater than 20-fold increase in bone morphogeneticprotein-2 (BMP-2) messenger RNA. BMP-2 is a member of the transforminggrowth factor (TGF) beta superfamily of proteins and a key regulator ofosteoblast differentiation. BMP is known to stimulate wound healing andincludes various bone morphogenetic proteins in addition to BMP-2. Thisalteration and increase of gene activity is indicative of the ability ofthe matrix to produce healing of bone fractures. This activity of thebioactive hydrogel matrix in stimulating BMP-2 production is illustratedin FIG. 5, which demonstrates an acute and dramatic increase in BMP-2gene expression after a 40 minute treatment with the bioactive hydrogelmatrix as compared to a control.

The useful activity of the bioactive hydrogel matrix is furtherdemonstrated in FIGS. 6 and 7, which illustrate the effects of treatmentof cultured chondrocytes (cells leading to the production of tendon,ligament, and cartilage) with the bioactive hydrogel matrix of theinvention in causing a greater than 3-fold increase in Connective TissueGrowth Factor (CTGF) and aggrecan gene expression. CTGF is a profibroticprotein induced by TGF beta and is a key regulator of chondrocyteproliferation and differentiation. It is an early marker ofchondrogenesis expressed at the highest levels in vivo duringchondrocyte growth. Aggrecan is a major cartilage extracellular matrix(ECM) component and a marker for the chondrocyte phenotype. FIG. 6 againillustrates an acute and marked increase in CTGF gene expression in thepresence of the bioactive hydrogel matrix. FIG. 7 illustrates a similarincrease in aggrecan gene expression and also illustrates a moreprolonged effect of such increase.

In addition to being in its usual, hydrated form (as generally describedabove), the bioactive hydrogel matrix of the present invention canfurther be in a dehydrated form. This is a particularly advantageousform of the bioactive hydrogel matrix increasing the practicalusefulness of the hydrogel matrix, providing for ease of storage andtransportation, and preserving the shelf-life of the hydrogel matrix andcompositions made using the hydrogel matrix. Any method generally knownin the art for dehydrating materials normally in a hydrated state wouldbe useful according to the present invention, so long as it is notdetrimental to the connective tissue regenerative properties of thehydrogel matrix as described herein. For example, one preferred methodof dehydrating the bioactive hydrogel matrix is freeze drying. Othermethods of preparing dehydrated biopolymers, such as spray-drying orspeed-vac, can also be used and are known to those skilled in the art.

Freeze drying generally comprises the removal of water or other solventfrom a frozen product through sublimation, which is the directtransition of a material (e.g., water) from a solid state to a gaseousstate without passing through the liquid phase. Freeze drying allows forthe preparation of a stable product being readily re-hydratable, easy touse, and aesthetic in appearance. The freeze drying process consists ofthree stages: 1) pre-freezing, 2) primary drying, and 3) secondarydrying.

Since freeze drying involves a phase change from solid to gaseous,material for freeze drying must first be adequately pre-frozen. Thepre-freezing method and the final frozen product temperature can bothaffect the ability to successfully freeze dry the material. Rapidcooling forms small ice crystals. While small crystals are useful inpreserving structure, they result in a product that is more difficult tofreeze dry. Slower cooling results in larger ice crystals and producesless restrictive channels in the matrix during the drying process.Pre-freezing to temperatures below the eutectic temperature, or glasstransition temperature, is necessary for complete drying of hydrogels.Inadequate freezing may produce small pockets of unfrozen materialremaining in the product which may expand and compromise the structuralstability of the freeze dried product.

After pre-freezing the product, conditions must be established in whichice (i.e., frozen solvent) can be removed from the frozen product viasublimation, resulting in a dry, structurally intact product. Thisrequires careful control of the two parameters, temperature andpressure, involved in the freeze drying system. It is important that thetemperature at which a product is freeze dried is balanced between thetemperature that maintains the frozen integrity of the product and thetemperature that maximizes the vapor pressure of the solvent.

After primary freeze drying is complete, and all ice has sublimed, boundmoisture is still present in the product. The product appears dry, butthe residual moisture content may be as high as 7-8%. Continued dryingis necessary at a warmer temperature to reduce the residual moisturecontent to optimum values. This process is called isothermal desorption,as the bound water is desorbed from the product. Secondary drying isnormally continued at a product temperature higher than ambient butcompatible with the sensitivity of the product. All other conditions,such as pressure and collector temperature, remain the same. Because theprocess is desorptive, the vacuum should be as low as possible (noelevated pressure) and the collector temperature as cold as can beattained. Secondary drying is usually carried out for approximately ⅓ to½ the time required for primary drying.

One example of equipment useful in preparing freeze dried hydrogels isthe FreeZone 12 Liter Freeze Dry System with Stoppering Tray Dryer(Labconco Kansas City, Mo.). With such system, tubes with porous capscontaining hydrogels are frozen to −30° C. at a cooling rate of 0.05°C./min using the cooling shelf unit of the freeze dryer and are held at−30° C. for 12 hours. A vacuum is applied to the frozen hydrogel at −30°C. for 24 hours before the temperature is incrementally increased to−10° C. at a rate of 0.25° C./minute. The hydrogel is held under vacuumat −10° C. for at least 12 hours before the temperature is furtherincreased to 20° C. at a rate of 0.05° C./minute.

The dehydrated bioactive hydrogel matrix can comprise the bioactivehydrogel matrix in any of the embodiments described herein. Furthermore,the bioactive hydrogel matrix can be used in preparing any of theconnective tissue regenerative compositions described herein prior tobeing dehydrated. Therefore, the present invention also encompassesdehydrated connective tissue regenerative compositions.

In one embodiment of the invention, the bioactive hydrogel matrix can beprepared as described herein and then dehydrated to form a single mass.The single mass can then be customized for specific uses. For example,the dehydrated hydrogel matrix could be sliced into wafer-like slices ofvarying dimensions. The dehydrated hydrogel matrix could also be groundto a particulate form. The dehydrated hydrogel matrix could also be cutto various shapes and dimensions for specified uses, such as pre-formedplugs for use in bone cavitation. Also, advantageously, the dehydratedbioactive hydrogel matrix could be formed to a standardized shape andsize and packaged for various uses. The pre-packaged dehydratedbioactive hydrogel matrix could then be customized to a desired shapeand size at the time of use. In a further embodiment, the dehydratedhydrogel matrix can be shaped around a central mandrel to form poroustubes useful for tissue regenerative guidance conduits. These can bewrapped around specific sites which may require or benefit from guidedtissue regeneration. Dehydrated hydrogels can also be partiallyrehydrated to form putties and pastes appropriate for filling bony voidscaused by surgery or trauma

The dehydrated hydrogel matrix, when re-hydrated, retains is connectivetissue regenerative properties as described herein and can be usedaccording to the methods of the invention as effectively as a freshlyprepared bioactive hydrogel matrix of the invention. The re-hydration ofthe hydrogel matrix can be performed according to various methods, allof which are encompassed by the invention. In one embodiment, thedehydrated bioactive hydrogel matrix is re-hydrated immediately prior touse, such as by contacting with water or a physiologically compatiblebuffer solution, such as Medium 199. In another embodiment, thedehydrated bioactive hydrogel matrix could be placed in the site in needof connective tissue regeneration and then contacted with re-hydratingfluids, such as water or a physiologically compatible buffer solution.In still another embodiment, the dehydrated hydrogel matrix could beplaced in the site in need of connective tissue regeneration and thenre-hydrated through contact with natural body fluids.

It is, of course, understood that any of the above embodiments describedin relation to the dehydrated hydrogel matrix are also intended toencompass similar or identical embodiments using the connective tissueregenerative compositions of the present invention comprising thebioactive hydrogel matrix.

While the bioactive hydrogel matrix of the present invention is usefulin multiple types of tissue repair, it is particularly advantageous inareas where tissue repair or regeneration is especially difficult. Asdescribed previously, such is often the case with bone regeneration andrepair of non-bone connective tissue. Connective tissue is a generalizedterm for mesodermally derived tissue that may be more or lessspecialized. Many types of tissue can fall under the term, such as bone,cartilage, dura mater, tendon, and ligament. The term can also be usedfor less specialized tissue that is rich in components such as collagenand proteoglycans, and that surrounds other more highly ordered tissuesand organs.

The bioactive hydrogel matrix is especially useful in the regenerationof bone, particularly in situations where bone repair does not occur orwhere more rapid healing of a bone defect would be beneficial to apatient. In situations where there is bone loss over of relatively largearea of the bone, the bioactive hydrogel matrix can be inserted into thearea of the bone loss and allowed to remain in place to facilitatehealing of the wound and regeneration of bone in the area of the loss.The matrix provides multiple regenerative functions as described above.The matrix interacts with osteocytes leading to more rapid formation ofbone tissue. The matrix also promotes osteoblast gene expression asdemonstrated by the increased production of BMP-2. The presence of thematrix in the wound site also inhibits ingrowth of non-bone tissue intothe wound inhibiting the formation of new bone tissue. The presence ofthe matrix also promotes vascularization, which is necessary for therapid growth of new bone tissue in providing nutrients, growth factors,oxygen, and other components necessary to bone regeneration.

Closely related to the ability of the matrix to promote regeneration ofbone is the function of the matrix in relation to stem or progenitorcells. This is an important aspect of the ability of the matrix tosupport tissue regeneration for multiple reasons. First, stem cells arefound in bone marrow, and these adult stem cells can be induced todifferentiate into bone tissue or other types of connective tissue,including cartilage, and important adjacent tissues, such as neurons andskeletal muscle. Further, progenitor cells, which are precursors givingrise to cells of a particular cell type, are also useful for inducingbone tissue growth, or other connective tissue growth, where applicable.Thus, interacting with those cells in the areas surrounding bone injury,for example, could stimulate stem or progenitor cells in the injuredarea to differentiate into bone cells, further hastening theregeneration of the bone. This is also significant in that often times,repair of hard tissue, such as bone, is accompanied by the need torepair soft tissue as well. One example is in the periodontal fieldwhere the presence of a material that would promote healing of the gumsas well as the underlying bone would be advantageous. A patient havingsevere periodontal disease with significant bone loss could be treatedusing the bioactive hydrogel matrix of the present invention. Thebioactive hydrogel matrix could be inserted into the area of bone lossand the gum tissue replaced over the area. The bioactive hydrogelmatrix, through its interaction with stem or progenitor cells andsubsequent changes in gene expression, as well as the other activitiesdescribed above, would not only facilitate the regeneration of the bone,but also hasten the repair of the gum tissue overlying the injured bone.The same type of action would be expected to take place in other typesof injury resulting in damage to bone as well as the surrounding tissue.

The present invention, in one aspect, is a method for regeneratingconnective tissue comprising administering a bioactive hydrogel matrixcomprising a polypeptide and a long chain carbohydrate, as describedherein, to a site in need of connective tissue regeneration.Preferentially, the polypeptide is a gelatin, and the long chaincarbohydrate is dextran.

The bioactive hydrogel matrix as used in the method of the invention caninclude one or more of the additional components previously notedherein. Additionally, the bioactive hydrogel matrix can incorporatefurther components facilitating the regeneration of connective tissueaccording to the method of the invention.

According to another aspect, the present invention provides variousconnective tissue regenerative compositions. Generally, the compositionscomprise a bioactive hydrogel matrix as described herein and at leastone additional component useful for accomplishing the methods of theinvention. Accordingly, any of the compositions described herein can beused in the various methods of the invention.

In one embodiment of the invention, the bioactive hydrogel matrixfurther comprises at least one osteoinductive or osteoconductivematerial. By “osteoinductive” is meant materials that lead to amitogenesis of undifferentiated perivascular mesenchymal cells leadingto the formation of osteoprogenitor cells (i.e., cells with the capacityto form new bone). By “osteoconductive” is meant materials thatfacilitate blood vessel incursion and new bone formation into a definedpassive trellis structure. Various compounds, minerals, proteins, andthe like are known to exhibit osteoinductive or osteoconductiveactivity. Accordingly, any of such materials would be useful accordingto the present invention.

In particular, any of the following could be used for theirosteoinductive or osteoconductive ability according to the presentinvention: demineralized bone matrix (DBM), bone morphogenetic proteins(BMPs), transforming growth factors (TGFs), fibroblast growth factors(FGFs), insulin-like growth factors (IGFs), platelet-derived growthfactors (PDGFs), epidermal growth factors (EGFs), vascular endothelialgrowth factors (VEGFs), vascular permeability factors (VPFs), celladhesion molecules (CAMs), calcium aluminate, hydroxyapatite, corallinehydroxyapatite, alumina, zirconia, aluminum silicates, calciumphosphate, tricalcium phosphate, calcium sulfate, polypropylenefumarate, bioactive glass, porous titanium, porous nickel-titaniumalloy, porous tantalum, sintered cobalt-chrome beads, ceramics,collagen, autologous bone, allogenic bone, xenogenic bone, coralline,and derivates or combinations thereof, or other biologically producedcomposite materials containing calcium or hydroxyapatite structuralelements.

By “alumina” is meant the commonly held definition of materialscomprised of the natural or synthetic oxide of aluminum, which may beexemplified in various forms, such as corundum. Bioactive glassesgenerally contain silicon dioxide (SiO₂) as a network former and arecharacterized by their ability to firmly attach to living tissue.Examples of bioactive glasses available commercially and theirmanufacturers include Bioglass® (American Biomaterials Corp., USA, 45%silica, 24% calcium oxide (CaO), 24.5% disodium oxide (Na₂O), and 6%pyrophosphate (P₂O₅)), Consil® (Xeipon Ltd., UK), NovaBone® (AmericanBiomaterials Corp.), Biogran® (Orthovita, USA), PerioGlass®(Block DrugCo., USA), and Ceravital® (E. Pfeil & H. Bromer, Germany). Corglaes®(Giltech Ltd., Ayr, UK) represents another family of bioactive glassescontaining pyrophosphate rather than silicon dioxide as a networkformer. These glasses contain 42-49 mole % of P₂O₅, the remainder as10-40 mole % as CaO and Na₂O.

When present in the bioactive hydrogel matrix of the present invention,the osteoinductive or osteoconductive material is preferably present ata volume concentration of about 0.01 percent to about 90 percent basedupon the total volume of the connective tissue regenerative composition.Such concentration is further dependent upon the ability to formcompositions having suitable putty or paste-like properties. Preferably,the osteoinductive or osteoconductive material is present at a volumeconcentration of about 50 percent to about 80 percent, based upon thetotal volume of the connective tissue regenerative composition. In oneparticular embodiment, a composition according to the inventioncomprises a 75% volume/volume mixture of osteoinductive orosteoconductive material, such as calcium sulfate, and bioactivehydrogel matrix (e.g., 12 mL calcium sulfate to 4 mL hydrogel matrix).

As a connective tissue regenerative composition, the bioactive hydrogelmatrix and the osteoinductive or osteoconductive materials can bevariously combined. Preferably, the osteoinductive or osteoconductivematerials and the hydrogel matrix are in admixture, which can beaccording to any means generally known to one of skill in the art. Forexample, the bioactive hydrogel matrix could be prepared, and theosteoinductive or osteoconductive material (e.g., powdered calciumphosphate) could be poured into and mixed into the hydrogel matrix bymechanical mixing means. The mixture could be flowable or could besubstantially thickened to a putty or paste-like consistency. Accordingto another embodiment, the bioactive hydrogel matrix could be dehydratedand, preferentially, in particulate or pelletized form. The particulatedehydrated bioactive hydrogel matrix could be mixed with anosteoinductive or osteoconductive material to form a substantiallyuniform mixture. In particular, the osteoinductive or osteoconductivematerial could be in the form of a putty or paste, and the particulatedehydrated bioactive hydrogel matrix kneaded or otherwise mixed therein.

In yet another embodiment of the invention, the bioactive hydrogelmatrix of the invention can further comprise at least one medicamentuseful for treating patients having connective tissue damage or in needof connective tissue regeneration. The medicament can be any medicamentuseful in facilitating the healing and regenerative process. Suchmedicaments useful according to the invention include, but are notlimited to, antivirals, antibacterials, anti-inflammatories,immunosuppressants, analgesics, anticoagulants, and wound healingpromotion agents.

According to another embodiment of the invention, the bioactive hydrogelmatrix can further comprise stem or progenitor cells, such as ADAScells, which are known to be capable of differentiating into adipogenic,osteogenic, chondrogenic, and myogenic lineages. Accordingly, thepresence of stem or progenitor cells can be beneficial for stimulatingand increasing connective tissue regrowth, particularly bone andcartilage. Further, the presence of the stem or progenitor cells can bebeneficial for stimulating and increasing growth of surrounding tissue,providing support for the damaged connective tissue. Preferably, stem orprogenitor cells are present at a concentration of about 10,000 to about1,000,000 cells per ml of hydrogel matrix, more preferably about 50,000to about 750,000 cells per ml of hydrogel matrix, most preferably about100,000 to about 500,000 cells per ml of hydrogel matrix. In oneparticular embodiment, the final concentration is about 250,000 cellsper ml of hydrogel matrix. In a further embodiment of the invention, thebioactive hydrogel matrix includes stem cells and progenitor cells. In aparticularly preferred embodiment, the progenitor cells areosteoprogenitor cells.

In one particular embodiment, the bioactive hydrogel matrix could be ina particulate, dehydrated form and the particles mixed into a solutioncontaining stem or progenitor cells, such as ADAS or mesenchymal stemcells.

In another embodiment of the invention, the bioactive hydrogel matrixincludes a three-dimensional structural framework. As previously noted,the bioactive hydrogel matrix of the present invention becomes flowableat physiological temperatures. As such, it is beneficial, in certainembodiments, for the bioactive hydrogel matrix to include structuralcomponents. Preferentially, the bioactive hydrogel matrix is at leastpartially contained within the three-dimensional structural framework.Accordingly, the structural framework can take on various embodiments.

In one particular embodiment, the three-dimensional structural frameworkincludes a scaffold or cage-like structure at least partially containingthe bioactive hydrogel matrix. Such an embodiment is particularly usefulin areas of long segmental diaphyseal bone loss or bone cavitation, orin the spinal column. The scaffold or cage-like structure spans the areaof bone loss and encloses the bioactive hydrogel matrix within the areaof bone loss. For example, the three-dimensional structural frameworkcould be a cylindrical metal mesh, such as titanium mesh. Accordingly,the three-dimensional structural framework can include materials thatare non-bioreabsorbable (i.e., persist in the body in a virtuallyunchanged state or must be later removed). Advantageously, thethree-dimensional structural framework includes a bioreabsorbablematerial that persists in the body long enough to perform itsstructure-providing function, later being broken down through naturalbody processes or being incorporated into the newly formed bone. In oneparticular embodiment, the three-dimensional structural frameworkincludes calcium-containing or calcified materials easily incorporatedinto newly formed bone.

In another embodiment, the three-dimensional structural framework is atleast partially internal to the bioactive hydrogel matrix. In suchembodiments, the three-dimensional structural framework preferablycomprises a material capable of physically or chemically interactingwith the hydrogel matrix. Preferably, the three-dimensional structuralframework provides an array of structural formations for providingsupport and structure to the bioactive hydrogel matrix.

It is particularly advantageous that the three-dimensional structuralframework be a structure that provides support and simultaneouslyprovides a space, or network of spaces, for cellular infiltration. It isparticularly beneficial for the three-dimensional structural frameworkto include a porous structure, such as a collagen or gelatin sponge. Anycommercially available collagen sponge would be useful generally in thepresent invention. Examples of commercially available collagen spongesinclude the Avitene Ultrafoam™ collagen sponge (available from Davol,Inc., a subsidiary of C.R. Bard, Inc., Murray Hill, N.J.—availableonline at http://www.davol.com), DuraGen® collagen sponge (availablefrom Integra LifeSciences Corp., Plainsboro, N.J.), and Gelfoam®, agelatin based sponge (available from Pharmacia & Upjohn, Kalamazoo,Mich.). Ceramic foams such as those produced by Hi-Por Ceramics(Sheffield, UK), could also be used.

The three-dimensional structural framework can be a single unit havingan inherent three-dimensional structure. As such, the structuralframework can be shaped as desired to precisely fit into the site inneed of connective tissue regeneration. This is particularly beneficialin cases of long segmental diaphyseal bone loss or bone cavitation. Insuch cases, the structural framework can be precisely shaped and sizedto the bone loss segment or cavitation to fill the space. The bioactivehydrogel matrix is retained in the bone loss segment or cavitation foran extended time period to facilitate bone regeneration by being atleast partially contained within the structural framework. To furtherplacement of the bioactive hydrogel matrix, the structural framework,with the hydrogel matrix contained therein, can optionally be suturedinto place.

The three-dimensional structural framework can comprise variousmaterials useful for providing structure and support and having aninherent three dimensional structure. The three-dimensional structuralframework can be a structure substantially in the form as found innature, such as coralline or natural sponge. Further, thethree-dimensional structural framework can be a fabricated structuremade from materials not naturally exhibiting a three-dimensionalstructure but being formed into such a structure, such as, for example,sintered calcium phosphate. Similarly, the three-dimensional structuralframework can comprise one or more polymeric materials that have beenmade, through processing (such as casting, molding, or sintering), intoa three-dimensional structure, particularly having a series or networkof pores or cavities throughout the structure for allowing cellularinfiltration. Examples of various materials useful as, or in thepreparation of, a three-dimensional structural framework include, butare not limited to, metals, calcium salts, coralline, bioactive glass,sponges, ceramics, collagen, keratin, fibrinogen, alginate, chitosan,hyaluronan, and other biologically-derived polymers. Thethree-dimensional structural framework can also comprise degradable andnon-degradable polymers, such as those commonly used in tissueengineering applications. Exemplary non-degradable polymers includepolyethylene, poly(vinylidene fluoride), poly(tetrafluoroethylene),poly(vinyl alcohol), poly(hydroxyalkanoate), poly(ethyleneterephthalate), poly(butylene terephthalate), poly(methyl methacrylate),poly(hydroxyethyl methacrylate), poly(N-isopropylacrylamide),poly(dimethyl siloxane), polydioxanone, and polypyrrole. Exemplarydegradable polymers include poly(glycolic acid), poly(lactic acids),poly(ethylene oxides), poly(lactide-co-glycolides),poly(s-caprolactone), polyanhydrides, polyphosphazenes,poly(ortho-esters), and polyimides.

In one particularly preferred embodiment, the three-dimensionalstructural framework comprises a crosslinked hydrogel matrix.Particularly preferred is a crosslinked bioactive hydrogel matrixcomprising a polypeptide, such as gelatin, and a long-chaincarbohydrate, such as dextran. Published U.S. Patent Application No.2003/0232746, which is incorporated herein by reference in its entirety,describes a crosslinked bioactive hydrogel matrix, wherein the hydrogelmatrix of the present invention is further stabilized and imparted athree-dimensional type structure through crosslinking of the matrixcomponents. Such crosslinked bioactive hydrogel matrix is also describedin PCT Publication No. WO 03/072155, which is also incorporated hereinby reference in its entirety. Additionally, Published U.S. PatentApplication No. 2003/0232198 and PCT Publication No. WO 03/072157, bothof which are also incorporated herein by reference in their entirety,describe a stabilized bioactive hydrogel matrix as a surface coating.Such crosslinked hydrogel matrices are also useful in the variousadditional embodiments of the present invention as described herein.

An example of a crosslinked bioactive hydrogel matrix comprising dextranand gelatin is provided in FIG. 8 wherein dextran 20 is covalentlycrosslinked to gelatin 15 by linkages 70, thereby forming a crosslinkednetwork 50. The linkages 70 either result from reaction of functionalgroups on the gelatin 15 with functional groups on the dextran 20, orresult from reaction of a bifunctional crosslinker molecule with boththe dextran 20 and gelatin 15. As explained in greater detail below, onemethod of crosslinking gelatin and dextran is to modify the dextranmolecules 20, such as by oxidation, in order to form functional groupssuitable for covalent attachment to the gelatin 15. This stabilizedcross-linked bio active network 50 yields therapeutically useful gelsand pastes that are insoluble in physiologic fluids at physiologicaltemperatures.

Crosslinked hydrogel matrices as useful according to the presentinvention can be prepared by various methods. In one particularembodiment, one of the polypeptides and long chain carbohydrates ismodified to form reactive groups suitable for crosslinking. Forinstance, the dextran or other long chain carbohydrate component can bemodified, such as by oxidation, in order to cross-link with thepolypeptide component. One known reaction for oxidizing polysaccharidesis periodate oxidation. The basic reaction process utilizing periodatechemistry is well known and appreciated by those skilled in the art.Periodate oxidation is described generally in Affinity Chromatography: APractical Approach, Dean, et al., IRL Press, 1985 ISBN0-904147-71-1,which is incorporated by reference in its entirety. The oxidation ofdextran by the use of periodate-based chemistry is described in U.S.Pat. No. 6,011,008, which is herein incorporated by reference in itsentirety.

In periodate oxidation, polysaccharides may be activated by theoxidation of the vicinal diol groups. With long chain carbohydrates,such as dextran, this is generally accomplished through treatment withan aqueous solution of a salt of periodic acid, such as sodium periodate(NaIO₄), which oxidizes the sugar diols to generate reactive aldehydegroups (e.g. dialdehyde residues). This method is a rapid, convenientalternative to other known oxidation methods, such as those usingcyanogen bromide. Dextran activated by periodate oxidation may be storedat 4° C. for several days without appreciable loss of activity.

Long chain carbohydrate materials, such as dextran, activated in thismanner readily react with materials containing amino groups, such aspolypeptides, particularly gelatin, producing a crosslinked materialthrough the formation of Schiff s base links. A Schiff base is a namecommonly used to refer to the imine formed by the reaction of a primaryamine with an aldehyde or ketone. The aldehyde groups formed on thecellulosic surface react with most primary amines between pH values fromabout 4 to about 6. The Schiff's base links form between the dialdehyderesidues of the dextran and the free amino groups on the gelatin. Thecrosslinked product may subsequently be stabilized (i.e. formation ofstable amine linkages) by reduction with a borohydride, such as sodiumborohydride (NaBH₄) or sodium cyanoborohydride (NaBH₃CN). The residualaldehyde groups may be consumed with ethanolamine or other aminecontaining species to further modify the crosslinked matrix. Othermethods known to those skilled in the art may be utilized to providereactive groups on either one or both of the polypeptide and long-chaincarbohydrate.

In preparing crosslinked bioactive hydrogel matrices for use in thepresent invention, periodate chemistry is preferentially used withdextran to form a multifunctional polymer that can then react withgelatin and other components, such as polar amino acids, present duringthe manufacturing process. The periodate reaction leads to the formationof polyaldehyde polyglycans that are reactive with primary amines Forexample, polypeptides and long chain carbohydrates may form covalenthydrogel complexes that are colloidal or covalently crosslinked gels.Covalent bonding occurs between reactive groups of the dextran andreactive groups of the gelatin component. The reactive sites on thegelatin include amine groups provided by arginine, asparagine,glutamine, and lysine. These amine groups react with the aldehyde orketone groups on the dextran to form a covalent bond. These hydrogelscan be readily prepared at temperatures from about 34° C. to about 90°C. Additionally, the hydrogels can be prepared at a pH range of fromabout 5 to about 9, preferably from about 6 to about 8, and mostpreferably from about 7 to about 7.6.

By controlling the extent of dextran activation and the reaction time,one can produce stabilized biomimetic scaffolding materials of varyingviscosity and stiffness. By “biomimetic” is intended compositions ormethods imitating or simulating a biological process or product. Somebiomimetic processes have been in use for several years, such as theartificial synthesis of vitamins and antibiotics. More recently,additional biomimetic applications have been proposed, includingnanorobot antibodies that seek and destroy disease-causing bacteria,artificial organs, artificial arms, legs, hands, and feet, and variouselectronic devices. The biomimetic scaffolding materials of the presentinvention yield therapeutically useful gels and pastes that are stableat about 37° C., or body temperature. These gels are capable ofexpansion and/or contraction, but will not dissolve in aqueous solution.Accordingly, such biomimetic crosslinked hydrogel matrices areparticularly useful as a three-dimensional structural framework asdescribed herein. The crosslinked hydrogel matrix provides the structureand support required, remains stable and maintains its three-dimensionalstructure at physiological temperatures, and can benefically providemany of the same connective tissue regenerative properties of thenon-crosslinked bioactive hydrogel matrix of the invention.

As an alternate method for forming the crosslinked dextran/gelatinnetwork, a multifunctional crosslinking agent may be utilized as areactive moiety that covalently links the gelatin and dextran chains.Such bifunctional crosslinking agents may include glutaraldehyde,epoxides (e.g., bis-oxiranes), oxidized dextran, p-azidobenzoylhydrazide, N-[α-maleimidoacetoxy]succinimide ester, p-azidophenylglyoxal monohydrate, bis-[β-(4-azidosalicylamido)ethyl]disulfide,bis[sulfosuccinimidyl]suberate, dithiobis[succinimidyl proprionate,disuccinimidyl suberate, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride, and other bifunctional crosslinking reagents known tothose skilled in the art.

In another embodiment utilizing a crosslinking agent, polyacrylatedmaterials, such as ethoxylated (20) trimethylpropane triacrylate, may beused as a non-specific photo-activated crosslinking agent. Components ofan exemplary reaction mixture would include a thermoreversible hydrogelheld at 39° C., polyacrylate monomers, such as ethoxylated (20)trimethylpropane triacrylate, a photo-initiator, such as eosin Y,catalytic agents, such as 1-vinyl-2-pyrrolidinone, and triethanolamine.Continuous exposure of this reactive mixture to long-wavelength light(>498 nm) would produce a crosslinked hydrogel network.

As with the non-crosslinked bioactive hydrogel matrix of the invention,the crosslinked hydrogel matrix can further comprise various additionalcomponents (in addition to the polypeptide and long chain carbohydrate)to enhance the crosslinked matrix by providing further stability orfunctional advantages. Such additional components can include anycompound, especially polar compounds, that, when incorporated into thecrosslinked hydrogel matrix, enhance the hydrogel matrix by providingfurther stability or functional advantages.

Preferred additional components for use with the stabilized crosslinkedhydrogel matrix include polar amino acids, polar amino acid analoguesand derivatives, intact collagen, and divalent cation chelators.Suitable concentrations of each particular preferred additionalcomponent are the same as noted above in connection with the bioactivehydrogel matrix of the present invention. Polar amino acids, EDTA, andmixtures thereof, are particularly preferred. The additional componentscan be added to the hydrogel matrix composition before or during thecrosslinking of the polypeptide and long chain carbohydrate.

In one embodiment of the invention, the bioactive hydrogel matrix of theinvention is in a crosslinked form, the three-dimensional structuralframework thus being inherent to the hydrogel matrix itself. In anotherembodiment of the invention, the bioactive hydrogel matrix of theinvention is first prepared, and the three-dimensional structuralframework, in the form of a separate, crosslinked hydrogel matrix, asdescribed above, is added to the non-crosslinked hydrogel matrix. Suchaddition can include physical admixture of the two hydrogels to form acomposition comprising a bioactive hydrogel matrix of the invention anda three-dimensional structural framework, in the form of a crosslinkedhydro gel matrix. In one particular embodiment, dehydrated cross-linkedbioactive hydrogel matrix formulations can be used with thenon-crosslinked hydrogel matrix or for providing structural supportgenerally. In this embodiment, the non-crosslinked hydrogel matrix canbe mixed with dehydrated crosslinked hydrogel matrix in the form ofdisks, rods, cylinders, granules, or other suitable geometric forms.Such compositions provide additional support to the surrounding tissue,and increase the localized residence time of the non-crosslinkedhydrogel matrix

Various embodiments of the invention can also be combined, particularlyin preparing the various connective tissue regenerative compositions ofthe invention. For example, in one particular embodiment, the bioactivehydrogel matrix/three-dimensional structural framework composition couldalso include osteoinductive or osteoconductive materials, stem orprogenitor cells (such as ADAS or mesenchymal stem cells), ormedicaments as described herein. Furthermore, any of these combinationscould be used according to the methods of the invention.

The methods of the invention wherein the bioactive hydrogel matrixincludes a three-dimensional structural framework are intended toencompass situations wherein the three-dimensional structural frameworkis included with the bioactive hydrogel matrix prior to administrationof the composition. Further, the methods encompass situations whereinthe three-dimensional structural framework is included with thebioactive hydrogel matrix after administration of the bioactive hydrogelmatrix.

The methods and compositions of the invention as described herein areuseful in the repair and regeneration of connective tissue, particularlybone, cartilage, ligament, and the like. As such, the methods andcompositions of the invention are particularly useful in varioustreatments involving portions of the human body particularly susceptibleto connective tissue damage or degeneration.

According to one embodiment of the invention, the bioactive hydrogelmatrix can be used in the surgical attachment or reattachment of one ormore connective tissues. Because of the generally decreasedvascularization of connective tissue types, healing times related toinjury repair are typically long in duration. Administration of thebioactive hydrogel matrix to the injured site during repair of theconnective tissue damage can improve integration of the tissuesresulting in a much stronger repair and decreased healing time. This isparticularly true in the reattachment of tendon or ligament to bone.Similar effects would be expected in the repair of torn ligaments ortorn tendons.

Accordingly, the present invention provides a method for reattachingconnective tissues to one another. The method generally comprisescoating at least a portion of at least one of the connective tissueswith the bioactive hydrogel matrix according to any of the variousembodiments of the present invention, contacting the connective tissues,and, optionally, suturing the connective tissues together. Generally,suturing (or use of other attachment aids, such as staples, glues, andadhesive strips) is advisable when using the bioactive hydrogel matrixto maintain the connection between the connective tissues during healingand reduce occurrence of separation of the tissues prior to sufficientre-growth and reattachment of the connective tissues. In one particularembodiment, at least a portion of the bioactive hydrogel matrix can bein crosslinked form.

The improved repair/regeneration supplied by the bioactive hydrogelmatrix would be beneficial to a large number of patients. Whilerelatively moderate numbers of patients suffer from large areas of boneloss, the matrix could also be used to treat injuries that are verycommon in occurrence, such as a torn anterior cruciate ligament (ACL),rotator cuff injuries, damaged cartilage in knees and other joints, andother such injuries that would be readily obvious to one of ordinaryskill in the art.

The bioactive hydrogel matrix of the invention further would be expectedto be beneficial for improved performance of any bioreabsorbable tissueanchors. Accordingly, in a further embodiment of the invention, thebioactive hydrogel matrix, according to any of the various compositionsdescribed herein, could be used in a combination therapy with otherknown reattachment devices. For example a torn connective tissue, suchas a tendon, could be reattached using sutures, staples, glues, or thelike, with at least one piece of the torn connective tissue being coatedat the area of the tear with a bioactive hydrogel matrix of the presentinvention to facilitate re-growth of connective tissue at the injurysite and hasten reattachment of the connective tissue.

According to another embodiment of the invention, the matrix is usefulin treating degenerative diseases, such as osteoarthritis, of thenatural joint of a patient. Osteoarthritis is characterized bydegeneration of the articular cartilage, hypertrophy of bone at themargins, and changes in the synovial membrane of the affected joint.Treatment of the affected joint with the bioactive hydrogel matrix ofthe invention, especially prior to complete loss of cartilage in thejoint, can stabilize the progression of the degeneration and evenpromote repair/regeneration of cartilage within the joint andregeneration of marginal bone structure. Further, the bioactive hydrogelmatrix of the invention provides an effective, minimally invasivetreatment, the hydrogel matrix being capable of injection directly intothe affected joint.

In this embodiment of the invention, the bioactive hydrogel matrix canbe in many of the various compositions as provided herein.Advantageously, the bioactive hydrogel matrix further comprises one ormore medicaments, osteoinductive or osteoconductive materials, or stemor progenitor cells.

In yet another embodiment of the invention, the bioactive hydrogelmatrix is useful in promoting the healing and effectiveness ofartificial joint replacements. According to this embodiment, the matrixis inserted into the area surrounding the artificial joint, particularlywhere the artificial joint is in contact with the natural tissue of thepatient. The hydrogel matrix facilitates the integration of theartificial joint into the surrounding tissue promoting faster healing ofthe surgical site and greater duration of the effectiveness of theartificial joint.

Preferably, a therapeutic amount of the matrix of the invention isadministered to a patient suffering from connective tissue injury,particularly an injury to bone, cartilage, tendon, or ligament. Thepatient can be any animal, including mammals such as dogs, cats andhumans. The term “therapeutic amount” refers to the amount required topromote tissue repair/regeneration as evidenced by, for example, theformation of new bone tissue across an area previously lacking in bonetissue. The therapeutic amount will be primarily determined by the sizeand type of injury being treated. Typically, the volume of bioactivehydrogel matrix applied is about 1 to about 60 mL, but could be greater,especially in large injuries, such as, for example, an area of greatbone loss in a large bone, such as a femur. Preferably, the therapeuticamount is sufficient to provide a uniform scaffolding for cellularattachment and differentiation in the area where tissue regeneration isneeded. The non-crosslinked version of the hydrogel matrix is typicallywarmed to a temperature of about 35 to about 40° C. prior toadministration in order to liquefy the matrix.

The bioactive hydrogel matrix used according to the methods of thepresent invention for regenerating connective tissue may be comprisedsolely of the polypeptide and long chain carbohydrate as describedherein. Preferably, the hydrogel matrix may incorporate additionalcomponents, such as the polar amino acids, polar amino acid analoguesand derivatives, and cation chelators, as described above. Table 1 belowlists particularly preferred components of the matrix of the presentinvention along with suitable concentrations as well as preferredconcentrations for each component. Note that the concentrations listedin Table 1 for gelatin and dextran would also be suitable foralternative polypeptide and long chain carbohydrate components.Bioactive hydrogel matrices prepared having the preferred components andconcentrations provided in Table 1 would also be particularly suitablefor use in the preparation of any of the connective tissue regenerativecompositions as described herein.

TABLE 1 Component Concentration Range Preferred Concentration L-glutamicacid 2 to 60 mM 20 mM L-lysine 0.5 to 30 mM 5.0 mM Arginine 1 to 40 mM15 mM Gelatin 0.01 to 40 mM 0.75 mM L-cysteine 5 to 5000 μM 700 μML-alanyl-L-glutamine 0.001 to 1 mM 0.01 mM EDTA 0.01 to 10 mM 4 mMDextran 0.01 to 10 mM 0.1 mM Zinc 0.005 to 3 mM 0.03 mM

EXPERIMENTAL

The present invention is more fully illustrated by the followingexamples, which are set forth to illustrate the present invention andare not to be construed as limiting thereof.

Example 1 Matrix Preparation

In one embodiment, the bioactive hydrogel matrix was compounded to yielda final formulation as described above in Table 1. Modified Medium 199(2.282 L) was placed into a stirred beaker. To the beaker were addedL-cysteine, L-glutamic acid, L-lysine, L-alanyl-L-glutamine, and EDTA.While stirring, the solution was heated to 50° C. Next, dextran wasadded, followed by the addition of gelatin. NaOH (10%) was used toadjust the pH of the matrix solution to a final pH of 7.50±0.05.Finally, additional L-glutamic acid, L-arginine, and L-cysteine wereadded followed by the addition of zinc sulfate. The amounts of eachcomponent used were the amounts necessary to bring the finalconcentration of each component to the preferred concentration providedin Table 1.

Example 2 Effect of Bioactive Hydrogel Matrix on Critical Size Defect inBone

The in vivo effect of the matrix on bone repair was examined using thecritical size defect model in the rabbit ulna. A defect in a rabbit ulnawas created in which the length of the defect was purposefully made tobe three times the diameter of the bone, i.e., a 15 mm defect wascreated in each ulna (the diameter of the bone was approximately 4 mm).It is well documented in the literature that defects of this size willnot spontaneously heal (i.e., a critical size defect). Seven rabbits had15 mm defects surgically created in the ulna of each forelimb. Further,the periosteum of the radius parallel to the defect was scraped off. Ineach rabbit, the defect in one forelimb was treated with the bioactivehydrogel matrix, and the other was treated with a collagen sponge soakedwith the bioactive hydrogel matrix. The muscle surrounding the bonedefect was sutured closed and the limb tightly wrapped.

The forelimbs of the rabbit were x-rayed to document the size of thedefect, with follow-up X-rays taken at two-week intervals to 10 weeks oftotal testing. Micro CT scans and histological examination were alsoperformed at 10 weeks. Radiographs of defect sites were scored forcalcification on a 0 to 4 scale. Mineralization within the defecttreated with the bioactive hydrogel matrix alone in combination with thecollagen sponge was noted as early as two weeks after the procedure, andby six weeks, clear and dramatic mineralization was evident within thearea of removed bone. The bioactive hydrogel matrix in the collagensponge tended to increase calcification compared to the hydrogel matrixalone, but the differences were not statistically significant. New boneformation within the defects was confirmed by both micro CT scans andhistopathology performed at 10 weeks.

Example 3 Effect of Bioactive Hydrogel Matrix on Tendon Re-Growth andStrength

Four sheep had a 4 mm length of the central portion of the patellartendon removed from the point of attachment of the tibia to the patellaof one leg. A small block of the patella with the attached patellartendon was also removed. The contra-lateral leg served as the unoperatedcontrol. Two defects were filled with a collagen sponge (DuraGen®,Integra LifeSciences) infiltrated with the bioactive hydrogel matrix ofthe invention, and two defects were filled with a DuraGen® collagensponge infiltrated with saline. The implants were sutured into thepatellar tendon defect and surgical site was sutured closed. After 12weeks, the patellar tendons were removed for gross observation andmechanical testing for stiffness. The tendons treated with the bioactivehydrogel matrix appeared thicker than the control tendons. Further, thetendons treated with the bioactive hydrogel matrix had an averageincrease in stiffness over the control tendons of about 17.2% comparedto a decrease of 4.5% in stiffness of the 2 tendons treated with thecollagen sponge soaked with saline.

Example 4 BMP-2 Gene Expression in Presence of Bioactive Hydrogel Matrix

Human osteosarcoma cells were plated in T75 flasks, allowed to grow toconfluency, and then shifted to serum-free medium (SFM) for three days.At this point cultures were treated for 40 minutes at 37° C. with eitherthe bioactive hydrogel matrix of the invention or serum-free medium as acontrol. Cultures were rinsed and re-fed with serum-free medium andsampled over a subsequent 24 hour period for extraction of nucleicacids. Messenger RNA from these preparations was used to createcomplementary DNA using reverse transcription, and specific DNAsequences were amplified and quantified using real-time polymerase chainreaction methods.

In several replicate experiments, induction of messenger RNA for bonemorphogenetic protein-2 (BMP-2) was induced as much as 44-fold, with apeak response seen 2 hours after treatment with the bioactive hydrogelmatrix with a return to baseline levels in 9 hours. Controls retainedbasal expression of the BMP-2 message over the entire 24 hour samplingperiod.

Expression of BMP-2 protein was also measured in these cell culturesfollowing identical culture and hydrogel matrix treatment methods, withthe exception that serum-containing medium (SCM) was used to avoid lossof analyte by adsorption to culture surfaces. For these studies cultureswere sampled over a 3-day period following either 40 or 120-minutestreatment by removal of medium and snap freezing the culture to releasecell-associated protein for analysis. Resulting samples were analyzed byenzyme-linked immunosorbent assay (ELISA) using a commercial BMP-2detection kit (R&D Systems, Minneapolis, Minn.). In replicate studies,treatment with the bioactive hydrogel matrix increased BMP-2 proteinlevels over controls within 24 hours post-treatment and provided as muchas a 3-fold increase over controls by day 3. These data are presentedgraphically in FIG. 9. These data demonstrate that the rapid increase ingene expression described earlier (FIG. 5) leads to a sustained increasein BMP-2 protein production.

Example 5 BMP-2 Gene Expression in Presence of Crosslinked BioactiveHydrogel Matrix

Samples of crosslinked bioactive hydrogel matrix prepared with 1%oxidized dextran were cast into disks to fit into 24-well plates. Diskswere sterilized in alcohol and equilibrated to culture medium and SAOS-2human osteosarcoma cells were seeded at approximately 330,000 per disk.After allowing time for cell attachment, disks were transferred to newwells and sampled on days 1, 2, 3 and 6 for extraction of nucleic acidsand quantitative PCR measurement of transcripts for BMP-2. Controls wereseeded cells that settled past the disks and attached and grew on thebottom of the original wells used for disk seeding. These studies showeda rise in BMP-2 mRNA that reached a peak nearly 3.5-fold greater thaninitial levels by day 3 and remained elevated at day 6. The results aredisplayed graphically in FIG. 10.

Example 6 Preparation of Compositions Including Bioactive HydrogelMatrix and Orthopedic Materials

A putty containing 2.4 g calcium sulfate and 1.5 g demineralized bonematrix (DBM) was prepared and used to incorporate particulate dehydratedbioactive hydrogel matrix. After briefly hand-kneading the material,about 0.5 g of particulate dehydrated bioactive hydrogel matrix(equivalent to about 3 mL of fully hydrated hydrogel matrix) wasincorporated into the putty. The putty was mixed and kneaded touniformity, and the putty retained the ability to fill a bony defectcreated in a synthetic bone

In a second composition, an injectable calcium sulfate formulation wasprepared using the bioactive hydrogel matrix of the invention ratherthan the conventional water base (12 g calcium sulfate powder and 4 mLof the bioactive hydrogel matrix). The material had a prolonged set time(over 2 hours to solidify), and during the early stages of setting wasjudged to be suitable for filling a bony defect.

In a third composition, the bioactive hydrogel matrix was mixed with agranulated tricalcium phosphate ceramic to form a moldable putty easilypacked into a bony defect.

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of the teachings presented in the foregoing descriptions andassociated drawings. Therefore, it is to be understood that theinvention is not to be limited to the specific embodiments disclosed andthat modifications and other embodiments are intended to be includedwithin the scope of the appended claims. Although specific terms areemployed herein, they are used in a generic and descriptive sense onlyand not for purposes of limitation.

1. A method for regenerating connective tissue selected from bone,cartilage, ligament, and tendon, comprising administering a bioactivehydrogel matrix to a site in need of connective tissue regeneration,said bioactive hydrogel matrix comprising: a polypeptide; a long chaincarbohydrate; one or more components selected from the group consistingof polar amino acids, polar amino acid analogs or derivatives, divalentcation chelators, and combination thereof; and at least oneosteoinductive or osteoconductive material selected from the groupconsisting of hydroxyapatite, demineralized bone matrix (DBM), bonemorphogenetic proteins (BMPs), transforming growth factors (TGFs),fibroblast growth factors (FGFs), insulin-like growth factors (IGFs),platelet-derived growth factors (PDGFs), epidermal growth factors(EGFs), vascular endothelial growth factors (VEGFs), and vascularpermeability factors (VPFs).
 2. The method of claim 1, wherein thepolypeptide is a tissue-derived polypeptide derived from tissue selectedfrom the group consisting of collagens, gelatins, keratin, decorin,aggrecan, and glycoproteins or is a tissue-derived polypeptide derivedfrom extracts of tissue selected from the group consisting of submucosaltissues, arteries, vocal chords, pleura, trachea, bronchi, pulmonaryalveolar septa, ligaments, auricular cartilage, abdominal fascia, liver,kidney, neurilemma, arachnoid, dura mater, and pia mater.
 3. The methodof claim 1, wherein the polypeptide has a molecular mass of about 3,000to about 3,000,000 Da.
 4. The method of claim 1, wherein the long chaincarbohydrate is a polysaccharide or a sulfated polysaccharide.
 5. Themethod of claim 4, wherein the long chain carbohydrate is apolysaccharide selected from the group consisting of dextran, dextrin,heparan, heparin, hyaluronic acid, chondroitin, alginate, agarose,carageenan, amylopectin, amylose, glycogen, starch, cellulose, chitin,and chitosan, or the long chain carbohydrate is a sulfatedpolysaccharide selected from the group consisting of heparan sulfate,heparin sulfate chondroitin sulfate, dextran sulfate, dermatan sulfate,and keratan sulfate.
 6. The method of claim 1, wherein the long chaincarbohydrate has a molecular mass of about 2,000 to about 8,000,000 Da.7. The method of claim 1, wherein the hydrogel matrix comprises one ormore polar amino acids selected from the group consisting of tyrosine,cysteine, serine, threonine, asparagine, glutamine, aspartic acid,glutamic acid, arginine, lysine, histidine, and mixtures thereof.
 8. Themethod of claim 1, wherein the hydrogel matrix comprisesethylenediaminetetraacetic acid or a salt thereof.
 9. The method ofclaim 1, wherein the bioactive hydrogel matrix further comprises atleast one medicament selected from the group consisting of antivirals,antibacterials, anti-inflammatories, immunosuppressants, analgesics,anticoagulants, and healing promotion agents.
 10. The method of claim 1,wherein the bioactive hydrogel matrix further comprises cells selectedfrom the group consisting of stem cells, progenitor cells, and mixturesthereof.
 11. The method of claim 1, wherein, after said administeringstep, the bioactive hydrogel matrix is flowable and is at leastpartially contained within a three-dimensional structural framework. 12.The method of claim 11, wherein the three-dimensional structuralframework includes a bioreabsorbable material.
 13. The method of claim11, wherein the three-dimensional framework includes a material selectedfrom the group consisting of coralline, metals, sponge, bioactive glass,ceramics, calcium salts, collagen, keratin, fibrinogen, alginate,chitosan, allogenic bone, autologous bone, hyaluronan, polyethylene,poly(vinylidene fluoride), poly(tetrafluoroethylene), poly(vinylalcohol), poly(hydroxyalkanoate), poly(ethylene terephthalate),poly(butylene terephthalate), poly(methy 1 methacrylate),poly(hydroxyethyl methacrylate), poly(N-isopropylacrylamide),poly(dimethyl siloxane), polydioxanone, and polypyrrole, poly(glycolicacid), poly(lactic acids), poly(ethylene oxides),poly(lactide-co-glycolides), poly(s-caprolactone), polyanhydrides,polyphosphazenes, poly(ortha-esters), and polyimides, and combinationsthereof.
 14. The method of claim 11, wherein the three-dimensionalstructural framework comprises a metal cage or a collagen sponge. 15.The method of claim 1, wherein the bioactive hydrogel matrix is indehydrated form prior to said administering step.
 16. The method ofclaim 15, wherein the dehydrated bioactive hydrogel matrix is inparticulate form.
 17. The method of claim 16, wherein the at least oneosteoinductive or osteoconductive material is dispersed within thebioactive hydrogel matrix.
 18. The method of claim 1, wherein at least aportion of the bioactive hydrogel matrix is in crosslinked form, thelong chain carbohydrate being covalently crosslinked to the polypeptide.19. The method of claim 1, wherein the osteoinductive or osteoconductivematerial comprises hydroxyapatite.
 20. The method of claim 19, whereinthe osteoinductive or osteoconductive material comprises demineralizedbone matrix (DBM).
 21. The method of claim 1, wherein the at least oneosteoinductive or osteoconductive material is present at a concentrationof about 0.01 volume percent to about 90 volume percent, based upon thetotal volume of the hydrogel matrix.
 22. The method of claim 21, whereinthe at least one osteoinductive or osteoconductive material is presentat a concentration of about 50 volume percent to about 80 volumepercent, based upon the total volume of the hydrogel matrix.
 23. Themethod of claim 1, comprising administering the composition at a siteundergoing surgical attachment or reattachment of the connective tissue.24. The method of claim 1, comprising administering the composition at asite of a torn anterior cruciate ligament (ACL), a rotator cuff injury,or damaged cartilage in a joint.
 25. The method of claim 1, wherein thepolypeptide comprises gelatin.
 26. The method of claim 25, wherein thelong chain carbohydrate comprises dextran.
 27. The method of claim 26,wherein bioactive hydrogel matrix further comprises at least one polaramino acid or divalent cation chelator.
 28. A method of treating adegenerative disease of the natural joint of a patient comprisingadministering a bioactive hydrogel matrix to a joint of a patientsuffering from the degenerative disease, the bioactive hydrogel matrixcomprising a polypeptide, a long chain carbohydrate, and at least oneosteoinductive or osteoconductive material selected from the groupconsisting of hydroxyapatite, demineralized bone matrix (DBM), bonemorphogenetic proteins (BMPs), transforming growth factors (TGFs),fibroblast growth factors (FGFs), insulin-like growth factors (IGFs),platelet-derived growth factors (PDGFs), epidermal growth factors(EGFs), vascular endothelial growth factors (VEGFs), and vascularpermeability factors (VPFs).
 29. The method of claim 28, wherein thepolypeptide comprises gelatin.
 30. The method of claim 28, wherein thebioactive hydrogel matrix further comprises at least one polar aminoacid or divalent cation chelator.
 31. The method of claim 28, whereinthe degenerative disease is arthritis.
 32. The method of claim 31,wherein the arthritis is osteoarthritis.
 33. The method of claim 31,comprising administering the bioactive hydrogel matrix prior to completeloss of cartilage in the joint.
 34. The method of claim 28, wherein saidadministering comprises injecting the bioactive hydrogel matrix into thejoint.
 35. The method of claim 28, wherein, prior to said administeringstep, the bioactive hydrogel matrix is in a dehydrated form and whereinthe method further comprises rehydrating the dehydrated bioactivehydrogel matrix.
 36. A method of promoting integration of an artificialjoint into the surrounding tissue comprising administering a bioactivehydrogel matrix at the site of the artificial joint, the bioactivehydrogel matrix comprising a polypeptide, a long chain carbohydrate, andat least one osteoinductive or osteoconductive material selected fromthe group consisting of hydroxyapatite, demineralized bone matrix (DBM),bone morphogenetic proteins (BMPs), transforming growth factors (TGFs),fibroblast growth factors (FGFs), insulin-like growth factors (IGFs),platelet-derived growth factors (PDGFs), epidermal growth factors(EGFs), vascular endothelial growth factors (VEGFs), and vascularpermeability factors (VPFs).